The major human pathogen Chlamydia trachomatis is an obligate intracellular bacterium that spends its entire replicative growth cycle within cellular vacuoles called inclusions. The chlamydial inclusion plays critical roles in establishing and maintaining a favorable intracellular niche for Chlamydia, and through the outer inclusion membrane of this vacuole, chlamydiae secrete numerous effector molecules into the host cell. The identity, function, and host interaction targets of the majority of these secreted proteins are not known, in large part due to historical challenges associated with genetic manipulation of Chlamydia. Recent breakthroughs in Chlamydia genetics have removed some of these major obstacles. Capitalizing on these advances, we have developed a genetic?proteomic system for C. trachomatis that selectively labels proteins in close proximity to the inclusion membrane, enabling the isolation and identification of these proteins by mass spectrometry. This system allows highly specific interactive mapping of the inclusion membrane proteome, even early in infection, and therefore makes possible the discovery of the bacterial and host proteins that comprise this pathogenic compartment in situ. The primary goals of this proposal are to: (i) determine the Chlamydia and host proteins that are dynamically recruited to the inclusion membrane at three stages of vacuolar growth, and (ii) extend the development of this technique for the identification of host molecular targets at two additional intracellular microenvironments targeted by chlamydial secreted proteins? the host cytosol and the inclusion lumen. These efforts are expected to derive a detailed proteomic map of host and Chlamydia proteins that are specifically recruited to early and late-stage inclusion membranes. The ability of this technique to capture interacting proteins in situ represents a major improvement over previous proteomic studies. We expect that the rich data generated from this project will yield novel insight into the cellular pathways and molecular factors targeted by Chlamydia during infection, and will provide the technical and biological resources for decades of future research focused on characterizing the molecular mechanisms of targets identified by this project. Finally, we anticipate that this technology can be broadly translated to other bacterial systems for analogous investigations of their secreted effectors and host targets.